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Mammalian mitochondrial ribosomes evolved from bacterial ribosomes by reduction of ribosomal RNAs, increase of ribosomal protein content, and loss of guanine nucleotides. Guanine is the base most sensitive to oxidative damage. By systematically comparing high-quality, small ribosomal subunit RNA sequence alignments and solved 3D ribosome structures from mammalian mitochondria and bacteria, we deduce rules for folding a complex RNA with the remaining guanines shielded from solvent. Almost all conserved guanines in both bacterial and mammalian mitochondrial ribosomal RNA form guanine-specific, local or long-range, RNA–RNA or RNA–protein interactions. Many solvent-exposed guanines conserved in bacteria are replaced in mammalian mitochondria by bases less sensitive to oxidation. New guanines, conserved only in the mitochondrial alignment, are strategically positioned at solvent inaccessible sites to stabilize the ribosomal RNA structure. New mitochondrial proteins substitute for truncated RNA helices, maintain mutual spatial orientations of helices, compensate for lost RNA–RNA interactions, reduce solvent accessibility of bases, and replace guanines conserved in bacteria by forming specific amino acid–RNA interactions.

Pyrene dihydrodioxins (PDHD) comprise effective DNA intercalation agents that are masked ortho-quinones, which can be released by near ultraviolet or visible irradiation. We have studied the binding and photoreactions of chiral dipyridinium PDHDs with herring sperm DNA and an 11-mer duplex DNA containing all 10 basepair steps. Binding affinities to herring sperm DNA were determined for purified enantiomers (Kb = 1.6 ± 0.15 × 105 and 2.3 ± 0.2 × 105 M−1). UV-melting experiments using the 11-mer DNA revealed significant stabilization of duplex DNA, (ΔTm = 11.5° and ΔTm = 15.3° C). Both enantiomers linearized (double-strand cleavage) supercoiled ΦX174 plasmid DNA with high efficiency. PDHDs have specificity to cleave and/or damage DNA duplexes at Gs and have preferable binding to GG DNA sites. A full range of transient absorption spectroscopy from the ultrafast femtosecond to the microsecond domains has been applied in the study of this system. These studies have revealed a novel mechanism for quinone release via the pyrene radical cation and the entrapment of the released electron by the coordinated action of the two pyridinium rings. These same studies have shown that the released pyrenequinone can photochemically initiate the further release of pyrenequinone. Thus, this reaction is autocatalytic and can be initiated with visible light.

RNA secondary structure diagrams familiar to molecular biologists summarize at a glance the folding of RNA chains to form Watson–Crick paired double helices. However, they can be misleading: First of all, they imply that the nucleotides in loops and linker segments, which can amount to 35% to 50% of a structured RNA, do not significantly interact with other nucleotides. Secondly, they give the impression that RNA molecules are loosely organized in three-dimensional (3D) space. In fact, structured RNAs are compactly folded as a result of numerous long-range, sequence-specific interactions, many of which involve loop or linker nucleotides. Here, we provide an introduction for students and researchers of RNA on the types, prevalence, and sequence variations of inter-nucleotide interactions that structure and stabilize RNA 3D motifs and architectures, using Escherichia coli (E. coli) 16S ribosomal RNA as a concrete example. The picture that emerges is that almost all nucleotides in structured RNA molecules, including those in nominally single-stranded loop or linker regions, form specific interactions that stabilize functional structures or mediate interactions with other molecules. The small number of noninteracting, ‘looped-out’ nucleotides make it possible for the RNA chain to form sharp turns. Base-pairing is the most specific interaction in RNA as it involves edge-to-edge hydrogen bonding (H-bonding) of the bases. Non-Watson–Crick base pairs are a significant fraction (30% or more) of base pairs in structured RNAs.

High affinity and specificity RNA–RNA binding interfaces can be constructed by combining pairs of GNRA loop/loop–receptor interaction motifs. These interactions can be fused using flexible four-way junction motifs to create divalent, self-assembling scaffolding units (‘tecto-RNA’) that have favorable properties for nanomedicine and other applications. We describe the design and directed assembly of tecto-RNA units ranging from closed, cooperatively assembling ring-shaped complexes of programmable stoichiometries (dimers, trimers and tetramers) to open multimeric structures. The novelty of this work is that tuning of the stoichiometries of self-assembled complexes is achieved by precise positioning of the interaction motifs in the monomer units rather than changing their binding specificities. Structure-probing and transmission electron microscopy studies as well as thermodynamic analysis support formation of closed cooperative complexes that are highly resistant to nuclease digestion. The present designs provide two helical arms per RNA monomer for further functionalization aims.

Motivation: Comparing 3D structures of homologous RNA molecules yields information about sequence and structural variability. To compare large RNA 3D structures, accurate automatic comparison tools are needed. In this article, we introduce a new algorithm and web server to align large homologous RNA structures nucleotide by nucleotide using local superpositions that accommodate the flexibility of RNA molecules. Local alignments are merged to form a global alignment by employing a maximum clique algorithm on a specially defined graph that we call the ‘local alignment’ graph. Results: The algorithm is implemented in a program suite and web server called ‘R3D Align’. The R3D Align alignment of homologous 3D structures of 5S, 16S and 23S rRNA was compared to a high-quality hand alignment. A full comparison of the 16S alignment with the other state-of-the-art methods is also provided. The R3D Align program suite includes new diagnostic tools for the structural evaluation of RNA alignments. The R3D Align alignments were compared to those produced by other programs and were found to be the most accurate, in comparison with a high quality hand-crafted alignment and in conjunction with a series of other diagnostics presented. The number of aligned base pairs as well as measures of geometric similarity are used to evaluate the accuracy of the alignments. Availability: R3D Align is freely available through a web server http://rna.bgsu.edu/R3DAlign. The MATLAB source code of the program suite is also freely available for download at that location. Supplementary information: Supplementary data are available at Bioinformatics online. Contact: r-rahrig@onu.edu

aptamers; biosensors; fluorescence; paranemic; RNA recognition; tokenRNAFluorescent reporters are highly sensitive, nonperturbing, and convenient probes for biologicalstudies. Current techniques for recognition of specific nucleic acid sequences usually requirecomplementary hybridization to chemically modified probes.[1] Optimal fluorescentbiosensors should rapidly signal the presence of a specific analyte with high selectivity andhigh contrast. Recently, a number of label-free, fluorescent bio-indicators have been reportedincluding aptamer-based riboswitches as sensors for cofactors,[2] aptamer-based proteinsensors,[3] and chimeric aptamers, in which the recognition domain binding the target wascoupled to an aptamer that binds a fluorophore in such a way that the presence of the targetincreases the affinity of the bioindicator for the fluorophore.[ 4,5] When the emission yields ofthe fluorophore in its bound and free states differ, this provides a binary sensor withnonperturbing optical recognition.It is desirable to detect macromolecular analytes (for example, structured RNA molecules) intheir native environment without having to denature or unfold them. In this work, wedemonstrate a technique for sensitive, label-free, real-time sequence-specific recognition ofprefolded RNA sequences. We use RNA constructs designed to form an aptameric pocket forthe fluorophore upon programmable paranemic binding to a specific prefolded analyte RNAsequence. We call these constructs paranemic “token RNAs”.We chose the triphenylmethane dye, Malachite Green (MG), as the fluorescent reporter becausein its unbound state in water solution it exhibits extremely low fluorescence quantum yieldfrom the S1 excited state because of efficient internal conversion.[ 6,7] The emission of the dyeincreases substantially when the nonradiative relaxation channels from S1 are shut down.Whereas the detailed underlying mechanisms of this phenomenon are still being debated,[7–9] related studies show that “rigidifying” the dye by placing it in a highly viscous environmentor in a binding cage increases its emission dramatically.[ 7] For instance, it was reported recentlythat the emission of MG increases by several orders of magnitude upon binding to an RNAaptamer obtained by in vitro selection (SELEX).[8,10] This aptamer has also been used as areporter for ATP recognition[4] and to perform real-time fluorescent monitoring of single-stranded DNA molecules.[11] The DNA detection was based on the separation of the MGaptamer into two strands, each of which is linked to a nucleic acid arm complementary to one

New RNA interaction interfaces are reported for designing RNA modules for directional supramolecular self-assembly. The new interfaces are generated from existing ones by inserting C-loops between the interaction motifs that mediate supramolecular assembly. C-Loops are new modular motifs recently identified in crystal structures that increase the helical twist of RNA helices in which they are inserted and thus reduce the distance between pairs of loop or loop-receptor motifs from 11 to 9 base-stacking layers while maintaining correct orientation for binding to cognate interaction interfaces. Binding specificities of C-loop-containing molecules for cognate molecules that also have inserted C-loops were found to range up to 20-fold. Binding affinities for most C-loop-containing molecules were generally equal or higher than those for the parent molecules lacking C-loops.

We present a comprehensive structural, evolutionary and molecular dynamics (MD) study of the G/U wobble basepairs in the ribosome based on high-resolution crystal structures, including the recent Escherichia coli structure. These basepairs are classified according to their tertiary interactions, and sequence conservation at their positions is determined. G/U basepairs participating in tertiary interactions are more conserved than those lacking any interactions. Specific interactions occurring in the G/U shallow groove pocket--like packing interactions (P-interactions) and some phosphate backbone interactions (phosphate-in-pocket interactions)--lead to higher G/U conservation than others. Two salient cases of unique phylogenetic compensation are discovered. First, a P-interaction is conserved through a series of compensatory mutations involving all four participating nucleotides to preserve or restore the G/U in the optimal orientation. Second, a G/U basepair forming a P-interaction and another one forming a phosphate-in-pocket interaction are replaced by GNRA loops that maintain similar tertiary contacts. MD simulations were carried out on eight P-interactions. The specific GU/CG signature of this interaction observed in structure and sequence analysis was rationalized, and can now be used for improving sequence alignments.

Molecular dynamics simulations of the frame-shifting pseudoknot from beet western yellows virus (BWYV, NDB file UR0004) were performed with explicit inclusion of solvent and counterions. In all, 33 ns of simulation were carried out, including 10 ns of the native structure with protonation of the crucial cytosine residue, C8(N3+). The native structure exhibited stable trajectories retaining all Watson-Crick and tertiary base-pairs, except for fluctuations or transient disruptions at specific sites. The most significant fluctuations involved the change or disruption of hydrogen-bonding between C8(N3+) and bases G12, A25, and C26, as well as disruption of the water bridges linking C8(N3+) with A25 and C26. To increase sampling of rare events, the native simulation was continued at 400 K. A partial, irreversible unfolding of the molecule was initiated by slippage of C8(N3+) relative to G12 and continued by sudden concerted changes in hydrogen-bonding involving A23, A24, and A25. These events were followed by a gradual loss of stacking interactions in loop 2. Of the Watson-Crick base-pairs, only the 5'-terminal pair of stem 1 dissociated at 400 K, while the trans sugar-edge/sugar-edge A20.G4 interaction remained surprisingly stable. Four additional room-temperature simulations were carried out to obtain insights into the structural and dynamic effects of selected mutations. In two of these, C8 was left unprotonated. Considerable local rearrangements occurred that were not observed in the crystal structure, thus confirming N3-protonation of C8 in the native molecule. We also investigated the effect of mutating C8(N3+) to U8, to correlate with experimental and phylogenetic studies, and of changing the G4 x C17 base-pair to A4 x U17 to weaken the trans sugar-edge interaction between positions 4 and 20 and to test models of unfolding. The simulations indicate that the C8 x G12 x C26 base-triple at the junction is the most labile region of the frame-shifting pseudoknot. They provide insights into the roles of the other non-Watson-Crick base-pairs in the early stages of unfolding of the pseudoknot, which must occur to allow readthrough of the message by the ribosome. The simulations revealed several critical, highly ordered hydration sites with close to 100 % occupancies and residency times of individual water molecules of up to 5 ns. Sodium cation coordination sites with occupancies above 50 % were also observed.

Bulges are common structural motifs in RNA secondary structure and are thought to play important roles in RNA-protein and RNA-drug interactions. Adenosine bases are the most commonly occurring unpaired base in double helical RNA secondary structures. The solution conformation and dynamics of a 25-nucleotide RNA duplex containing an unpaired adenosine, r(GGCAGAGUGCCGC): r(GCGGCACCUGCC) have been studied by NMR spectroscopy and MORASS iterative relaxation matrix structural refinement. The results show that the bulged adenosine residue stacks into the RNA duplex with little perturbation around the bulged region. Most of the bases in the RNA duplex adopt C3′-endo conformation, exhibiting the N-type sugar pucker as found in the A form helices. The sugars of the bulged residue and the 5′ flanking residue to it are found to exhibit C2′-endo conformation. None of the residues are in syn conformation.

Homonuclear 3D NOESY-NOESY has shown great promise for the structural refinement of large biomolecules. A computationally efficient hybrid-hybrid relaxation matrix refinement methodology, using 3D NOESY-NOESY data, was used to refine the structure of a DNA three-way junction having two unpaired bases at the branch point of the junction. The NMR data and the relaxation matrix refinement confirm that the DNA three-way junction exists in a folded conformation with two of the helical stems stacked upon each other. The third unstacked stem extends away from the junction, forming an acute angle (∼60° ) with the stacked stems. The two unpaired bases are stacked upon each other and are exposed to the solvent. Helical parameters for the bases in all three strands show slight deviations from typical values expected for right-handed B-form DNA. Inter-nucleotide imino-imino NOEs between the bases at the branch point of the junction show that the junction region is well defined. The helical stems show mobility (± 20° ) indicating dynamic processes around the junction region. The unstacked helical stem adjacent to the unpaired bases shows greater mobility compared to the other two stems. The results from this study indicate that the 3D hybrid-hybrid matrix MORASS refinement methodology, by combining the spectral dispersion of 3D NOESY-NOESY and the computational efficiency of 2D refinement programs, provides an accurate and robust means for structure determination of large biomolecules. Our results also indicate that the 3D MORASS method gives higher quality structures compared to the 2D complete relaxation matrix refinement method.

The sarcin-ricin RNA motif (SR motif) is one of the most prominent recurrent RNA building blocks that occurs in many different RNA contexts and folds autonomously, that is, in a context-independent manner. In this study, we combined bioinformatics analysis with explicit-solvent molecular dynamics (MD) simulations to better understand the relation between the RNA sequence and the evolutionary patterns of the SR motif. A SHAPE probing experiment was also performed to confirm the fidelity of the MD simulations. We identified 57 instances of the SR motif in a nonredundant subset of the RNA X-ray structure database and analyzed their base pairing, base-phosphate, and backbone-backbone interactions. We extracted sequences aligned to these instances from large rRNA alignments to determine the frequency of occurrence for different sequence variants. We then used a simple scoring scheme based on isostericity to suggest 10 sequence variants with a highly variable expected degree of compatibility with the SR motif 3D structure. We carried out MD simulations of SR motifs with these base substitutions. Nonisosteric base substitutions led to unstable structures, but so did isosteric substitutions which were unable to make key base-phosphate interactions. The MD technique explains why some potentially isosteric SR motifs are not realized during evolution. We also found that the inability to form stable cWW geometry is an important factor in the case of the first base pair of the flexible region of the SR motif. A comparison of structural, bioinformatics, SHAPE probing, and MD simulation data reveals that explicit solvent MD simulations neatly reflect the viability of different sequence variants of the SR motif. Thus, MD simulations can efficiently complement bioinformatics tools in studies of conservation patterns of RNA motifs and provide atomistic insight into the role of their different signature interactions.

Competition binding and UV melting studies of a DNA model system consisting of three, four or five mutually complementary oligonucleotides demonstrate that unpaired bases at the branch point stabilize three- and five-way junction loops but destabilize four-way junctions. The inclusion of unpaired nucleotides permits the assembly of five-way DNA junction complexes (5WJ) having as few as seven basepairs per arm from five mutually complementary oligonucleotides. Previous work showed that 5WJ, having eight basepairs per arm but lacking unpaired bases, could not be assembled [Wang, Y.L., Mueller, J.E., Kemper, B. and Seeman, N.C. (1991) Biochemistry, 30, 5667-5674]. Competition binding experiments demonstrate that four-way junctions (4WJ) are more stable than three-way junctions (3WJ), when no unpaired bases are included at the branch point, but less stable when unpaired bases are present at the junction. 5WJ complexes are in all cases less stable than 4WJ or 3WJ complexes. UV melting curves confirm the relative stabilities of these junctions. These results provide qualitative guidelines for improving the way in which multi-helix junction loops are handled in secondary structure prediction programs, especially for single-stranded nucleic acids having primary sequences that can form alternative structures comprising different types of junctions.

Three-arm DNA junctions, in which three double helices intersect at a branch, have unique structure and reactivity of bases at and near the branch. Their solution conformation is asymmetric in the presence of Mg2+, while bases at the branch are sensitive to single-strand-specific agents. Following the surprising report that unpaired bases at the branch stabilize three-arm junctions, we have investigated the geometry and thermodynamics of three-arm junctions containing pendant T and A bases. The results are consistent with additional structure formation in junctions containing up to four pendant bases at the branch: relative to the tight junction, the thermal stability of junctions with two T's or A's at the branch increases; bases near the branch become less reactive to single-strand-reactive probes; and the enthalpy of formation is more negative. The interaction of ethidium observed at the branch in three-arm junctions is enhanced in junctions with unpaired bases at the branch. The geometry of three-arm junctions is perturbed by the presence of pendant bases, as seen by measuring the electrophoretic mobility of junctions to which long duplex arms are appended pairwise.

RNA molecules are stabilized by a wide range of noncanonical interactions that are not present in DNA. Among them, the recently classified base-phosphate (BPh) interactions belong to the most important ones. Twelve percent of nucleotides in the ribosomal crystal structures are involved in BPh interactions. BPh interactions are highly conserved and provide major constraints on RNA sequence evolution. Here we provide assessment of the energetics of BPh interactions using MP2 computations extrapolated to the complete basis set of atomic orbitals and corrected for higher-order electron correlation effects. The reference computations are compared with DFT-D and DFT-D3 approaches, the SAPT method, and the molecular mechanics force field. The computations, besides providing the basic benchmark for the BPh interactions, allow some refinements of the original classification, including identification of some potential doubly bonded BPh patterns. The reference computations are followed by analysis of some larger RNA fragments that consider the context of the BPh interactions. The computations demonstrate the complexity of interaction patterns utilizing the BPh interactions in real RNA structures. The BPh interactions are often involved in intricate interaction networks. We studied BPh interactions of protonated adenine that can contribute to catalysis of hairpin ribozyme, the key BPh interaction in the S-turn motif of the sarcin-ricin loop, which may predetermine the S-turn topology and complex BPh patterns from the glmS riboswitch. Finally, the structural stability of BPh interactions in explicit solvent molecular dynamics simulations is assessed. The simulations well preserve key BPh interactions and allow dissection of structurally/functionally important water-meditated BPh bridges, which could not be considered in earlier bioinformatics classification of BPh interactions.

Biomedical Ontologies are intended to integrate diverse biomedical data to enable intelligent data-mining and facilitate translation of basic research into useful clinical knowledge. We present the first version of RNAO, an ontology for integrating RNA 3D structural, biochemical and sequence data. While each 3D data file depicts the structure of a specific molecule, such data have broader significance as representatives of classes of homologous molecules, which, while differing in sequence, generally share core structural features of functional importance. Thus, 3D structure data gain value by being linked to homologous sequences in genomic data and databases of sequence alignments. Likewise genomic data can increase in value by annotation of shared structural features, especially when these can be linked to specific functions. The RNAO is being developed in line with the developing standards of the Open Biomedical Ontologies (OBO) Consortium.

Structured RNA molecules form complex 3D architectures stabilized by multiple interactions involving the nucleotide base, sugar and phosphate moieties. A significant percentage of the bases in structured RNA molecules in the Protein Data Bank (PDB) hydrogen-bond with phosphates of other nucleotides. By extracting and superimposing base-phosphate (BPh) interactions from a reduced-redundancy subset of 3D structures from the PDB, we identified recurrent phosphate-binding sites on the RNA bases. Quantum chemical calculations were carried out on model systems representing each BPh interaction. The calculations show that the centers of each cluster obtained from the structure superpositions correspond to energy minima on the potential energy hypersurface. The calculations also show that the most stable phosphate-binding sites occur on the Watson–Crick edge of guanine and the Hoogsteen edge of cytosine. We modified the ‘Find RNA 3D' (FR3D) software suite to automatically find and classify BPh interactions. Comparison of the 3D structures of the 16S and 23S rRNAs of Escherichia coli and Thermus thermophilus revealed that most BPh interactions are phylogenetically conserved and they occur primarily in hairpin, internal or junction loops or as part of tertiary interactions. Bases that form BPh interactions, which are conserved in the rRNA 3D structures are also conserved in homologous rRNA sequence alignments.

Non-paired nucleotides stabilize the formation of three-way helical DNA junctions. Two or more unpaired nucleotides located in the junction region enable oligomers ten to fifteen nucleotides long to assemble, forming conformationally homogeneous junctions, as judged by native gel electrophoresis. The unpaired bases can be present on the same strand or on two different strands. Up to five extra bases on one strand have been tested and found to produce stable junctions. The formation of stable structures is favored by the presence of a divalent cation such as magnesium and by high monovalent salt concentration. The order-disorder transition of representative three-way junctions was monitored optically in the ultraviolet and analyzed to quantify thermodynamically the stabilization provided by unpaired bases in the junction region. We report the first measurements of the thermodynamics of adding an unpaired nucleotide to a nucleic acid three-way junction. We find that delta delta G degrees (37 degrees C) = +0.5 kcal/mol for increasing the number of unpaired adenosines from two to three. Three-way junctions having reporter arms 40 base-pairs long were also prepared. Each of the three reporter arms contained a unique restriction site 15 base-pairs from the junction. Asymmetric complexes produced by selectively cleaving each arm were analyzed on native gels. Cleavage of the double helical arm opposite the strand having the two extra adenosines resulted in a complex that migrated more slowly than complexes produced by cleavage at either of the other two arms. It is likely that the strand containing the unpaired adenosines is kinked at an acute angle, forming a Y-shaped, rather than a T-shaped junction.

Photoinduced electron transfer between cytochrome c and free base and metallouroporphyrin (Up, MUp) has been studied. Difference absorption spectrophotometry showed that the electrostatic interactions between Up and cytc(III) result in their forming a self-associated 1:1 complex in the ground state with a binding constant that depends upon the ionic strength. In the complex, the photoexcited uroporphyrin singlet state was quenched through a static interaction with the protein. Even under the most favorable quenching conditions, i.e., when all porphyrin was complexed, residual fluorescence was noted. More significantly the excited singlet state of the complex was shown to undergo small, but significant, intersystem crossing. These triplet states rapidly underwent an electron-transfer process that yielded transiently the Fe(II) form of the protein. This is the first observation of such a process from a porphyrin/cytochrome self-association complex.

The paranemic crossover (PX) is a motif for assembling two nucleic acid molecules using Watson-Crick (WC) basepairing without unfolding preformed secondary structure in the individual molecules. Once formed, the paranemic assembly motif comprises adjacent parallel double helices that crossover at every possible point over the length of the motif. The interaction is reversible as it does not require denaturation of basepairs internal to each interacting molecular unit. Paranemic assembly has been demonstrated for DNA but not for RNA and only for motifs with four or more crossover points and lengths of five or more helical half-turns. Here we report the design of RNA molecules that paranemically assemble with the minimum number of two crossovers spanning the major groove to form paranemic motifs with a length of three half turns (3HT). Dissociation constants (Kd's) were measured for a series of molecules in which the number of basepairs between the crossover points was varied from five to eight basepairs. The paranemic 3HT complex with six basepairs (3HT_6M) was found to be the most stable with Kd = 1 x 10-8 M. The half-time for kinetic exchange of the 3HT_6M complex was determined to be approximately 100 min, from which we calculated association and dissociation rate constants ka = 5.11 x 103 M-1s-1 and kd = 5.11 x 10-5 s-1. RNA paranemic assembly of 3HT and 5HT complexes is blocked by single-base substitutions that disrupt individual intermolecular Watson-Crick basepairs and is restored by compensatory substitutions that restore those basepairs. The 3HT motif appears suitable for specific, programmable, and reversible tecto-RNA self-assembly for constructing artificial RNA molecular machines.

Molecular dynamics simulations of RNA-protein complex between Escherichia coli loop E/helix IV (LE/HeIV) rRNA and L25 protein reveal a qualitative agreement between the experimental and simulated structures. The major groove of LE is a prominent rRNA cation-binding site. Divalent cations rigidify the LE major groove geometry whereas in the absence of divalent cations LE extensively interacts with monovalent cations via inner-shell binding. The HeIV region shows bistability of its major groove explaining the observed differences between x-ray and NMR structures. In agreement with the experiments, the simulations suggest that helix-α1 of L25 is the least stable part of the protein. Inclusion of Mg2+ cations into the simulations causes perturbation of basepairing at the LE/HeIV junction, which does not, however, affect the protein binding. The rRNA-protein complex is mediated by a number of highly specific hydration sites with long-residing water molecules and two of them are bound throughout the entire 24-ns simulation. Long-residing water molecules are seen also outside the RNA-protein contact areas with water-binding times substantially enhanced compared to simulations of free RNA. Long-residency hydration sites thus represent important elements of the three-dimensional structure of rRNA.

X-ray, phylogenetic and quantum chemical analysis of molecular interactions and conservation patterns of cis Watson-Crick (W.C.) A/G base-pairs in 16S rRNA, 23S rRNA and other molecules was carried out. In these base-pairs, the A and G nucleotides interact with their W.C. edges with glycosidic bonds oriented cis relative to each other. The base-pair is stabilised by two hydrogen bonds, the C1'-C1' distance is enlarged and the G(N2) amino group is left unpaired. Quantum chemical calculations show that, in the absence of other interactions, the unpaired amino group is substantially non-planar due to its partial sp(3) pyramidalization, while the whole base-pair is internally propeller twisted and very flexible. The unique molecular properties of the cis W.C. A/G base-pairs make them distinct from other base-pairs. They occur mostly at the ends of canonical helices, where they serve as interfaces between the helix and other motifs. The cis W.C. A/G base-pairs play crucial roles in natural RNA structures with salient sequence conservation patterns. The key contribution to conservation is provided by the unpaired G(N2) amino group that is involved in a wide range of tertiary and neighbor contacts in the crystal structures. Many of them are oriented out of the plane of the guanine base and utilize the partial sp(3) pyramidalization of the G(N2). There is a lack of A/G to G/A covariation, which, except for the G(N2) position, would be entirely isosteric. On the contrary, there is a rather frequent occurrence of G/A to G/U covariation, as the G/U wobble base-pair has an unpaired amino group in the same position as the cis W.C. G/A base-pair. The cis W.C. A/G base-pairs are not conserved when there is no tertiary or neighbor interaction. Obtaining the proper picture of the interactions and phylogenetic patterns of the cis W.C. A/G base-pairs requires a detailed analysis of the relation between the molecular structures and the energetics of interactions at a level of single H-bonds and contacts.

Cationic porphyrins form ground state electrostatically associated complexes with anionic oligo-electrolytes such as those formed by a series of glutamic acid (E) residues. Temperature dependencies were measured of the rate constants for intra-complex electron transfer to the triplet state of Pd(II)TMPyP4+ from a tyrosine (tyr, Y) or tryptophan (trp, W) moiety connected to a glutamic acid tetramer. In complexes such as YE4, E2YE2, YE4G10E (G, glycine), and WE4 these data were used to estimate the reorganization energy (lambda) and electronic interaction energy (HDA) relevant to the process. For all tyr-peptide complexes, lambda values were found to be large (lambda approximately 1.60 +/- 0.06 eV), reflecting a relatively high medium polarity in the vicinity of tyr residues. It further indicates that the tyr residues in all oligo-peptides are exposed to the aqueous medium in a similar way irrespective of the position of the aromatic moiety in the peptide chain. A significantly lower lambda value (lambda = 1.08 eV) was derived for the tryptophan-containing peptide complex, indicating a relatively higher hydrophobic character of trp compared to tyr. The electronic coupling matrix elements (HDA) derived for tyr-peptide complexes (5.1 meV for YE4, 5.4 meV for YE4G10E and 7.5 meV for E2YE2) were larger than that found for WE4 (1.1 meV). Molecular dynamics calculations were employed to obtain structural features of the porphyrin-peptide complexes. These showed average distances between the center of mass (COM) of the porphyrin ring and the center of mass of the amino acid aromatic ring of 816 +/- 140 pm (YE4), 800 +/- 80 pm (E2YE2), 900 +/- 130 pm (YE4G10E) and 970 +/- 160 pm (WE4). The molecular dynamics calculations were shown to be in good agreement with the experimentally determined electronic interaction energies, strongly suggesting that HDA is primarily responsible for the dependence of the electron-transfer rate constant (KET) on the donor-acceptor separation distance and relative orientation. The higher HDA (7.55 meV) derived for tyr incorporated into the middle of the peptide backbone (E2YE2) was presumed to be associated with a higher degree of orbital overlap due to a more favorable ring-ring orientation. Overlap parameters (beta derived for all peptide-porphyrin complexes were similar (approximately 0.95 +/- 0.06 A-1), being in good agreement with most literature values for similar systems. Finally, the intra-complex electron-transfer ratio (ktrp/ktyr) derived from flash photolysis experiments and the corresponding ratio derived from Marcus' theory combined with experimental data from the temperature-dependence investigations and electrochemical measurements were found to be in excellent agreement. This same consistency was found for the couple E4Y and E2YE2. The empirical expression (Moser and Dutton) governing the intraprotein electron-transfer rate constant in native systems combined with our experimental data (kET, lambda, delta G0) yielded tunneling pathway distances in excellent agreement with those arising from the molecular modeling studies. The exception was for the long peptide YE4G10E, for which the Quenched Molecular Dynamic (QMD) sampling technique was complicated and is probably inadequate.

The interaction of 5,10,15, 20-tetrakis(N-methylpyridinium-4-yl)porphyrin (T4MPyP(4+)) with the oligonucleotide DNA duplex [d(GCACGTGC)](2) was studied by two-dimensional (1)H NMR spectroscopy, optical absorbance, circular dichroism, and molecular dynamics simulation employing particle mesh Ewald methods. T4MPyP(4+) is one of the largest aromatic molecules for which intercalative binding to DNA has been proposed, although this has been called into question by recent X-ray crystallographic work [Lipscomb et al. (1996) Biochemistry 35, 2818-2823]. T4MPyP(4+) binding to [d(GCACGTGC)](2) produced a single set of (mostly) upfield-shifted DNA resonances in slow exchange with the resonances of the free DNA. Intra- and intermolecular NOEs observed in the complex showed that the porphyrin intercalates at the central 5'-CG-3' step of the DNA duplex without disrupting the flanking base pairs. Absorption and circular dichroism spectra of the complex also support intercalative binding. Molecular dynamics simulations (using explicit solvent and PME methods), carried out for fully and partially intercalated complexes, yielded stable trajectories and plausible structures, but only the symmetrical, fully intercalated model agreed with NOESY data. Stable hydrogen bonding was observed during 600 ps of MD simulation for the base pairs flanking the binding site.

Single-stranded regions of structured RNAs often participate in complex interactions involving non-canonical basepairs. A dictionary of isosteric pairings that substitute for each other while preserving the 3D structure of conserved motifs can be inferred from phylogenetic analysis of conserved «internai loops» for which high-resolution structures also exist, such as loop E of 5S rRNA and the sarcin/ricin loop of 23S rRNA. Such «dictionaries» of isosteric, context-specific pairings allow one to identify occurrences of the same motif in other molecules.

The proton NMR spectra of DNA three-way junction complexes (TWJ) having unpaired pyrimidines, 5'-TT- and 5'-TC- on one strand at the junction site were assigned from 2D NOESY spectra acquired in H2O and D2O solvents and homonuclear 3D NOESY-TOCSY and 3D NOESY-NOESY in D2O solvent. TWJ are the simplest branched structures found in biologically active nucleic acids. Unpaired nucleotides are common features of such structures and have been shown to stabilize junction formation. The NMR data confirm that the component oligonucleotides assemble to form conformationally homogeneous TWJ complexes having three double-helical, B-form arms. Two of the helical arms stack upon each other. The unpaired pyrimidine bases lie in the minor groove of one of the helices and are partly exposed to solvent. The coaxial stacking arrangement deduced is different from that determined by Rosen and Patel (Rosen, M.A., and D.J. Patel. 1993. Biochemistry. 32:6576–6587) for a DNA three-way junction having two unpaired cytosines, but identical to that suggested by Welch et al. (Welch, J. B., D. R. Duckett, D. M. J. Lilley. 1993. Nucleic Acids Res. 21:4548–4555) on the basis of gel electrophoretic studies of DNA three-way junctions containing unpaired adenosines and thymidines.

Publisher Summary This chapter discusses the structural studies of DNA three-way junctions. Advances in the nuclear magnetic resonance (NMR) methods allow one to study increasingly complex molecular assemblages. The chapter describes the use of two- and three-dimensional homonuclear (proton) NMR, coupled with other experimental approaches, physical and chemical, to investigate the structure of DNA three-way junctions (TWJ) composed of three mutually complementary oligonucleotide strands. Synthetic TWJ oligonucleotide complexes serve as models of multibranch structures that occur frequently in biologically important nucleic acids, such as the ribosomal RNAs and the hammerhead ribozymes. Examination of the sequences, of naturally occurring TWJ, indicates that evolutionarily conserved unpaired bases are integral components of these structures. Using gel electrophoresis and UV melting techniques, the chapter shows that unpaired bases stabilize TWJ complexes. The TWJ that have been successfully studied by NMR have included two unpaired nucleotides in the junction region. The construction and refinement of the putative structural models based on the NMR data and the additional information obtained from chemical probing are also described in the chapter. Successful structure determination is greatly facilitated, by having at hand a molecular object, that exists in one predominant conformation or in a set of closely related conformational states.

Isothermal titration calorimetry (ITC) is used to study the thermodynamics of assembly of the three DNA oligonucleotides S1 (5'-GCCTGCCACCGC), S2 (5'-GCGGTGCGTCCG), and S3AA (5'-CGGACGAAGCAGGC) to form a three-way junction (TWJ) complex consisting of three double-helical arms radiating from a junction region having two unpaired adenosines in one strand (S3AA). The thermodynamics of assembly were measured for three different orders of addition of the component oligonucleotides at four temperatures between 10 and 25 degrees C. At each temperature studied, the overall values of delta H, delta S degrees, and delta G degrees for assembly of the complex from the component single strands were found to be independent of the order of addition. The enthalpy of binding, delta H, was found to be linearly dependent on temperature. From the temperature dependence of delta H, the change in heat capacity delta Cp, for the overall assembly of three strands to form the junction complex was calculated and found to be -1.6 kcal mol-1K-1. This work represents the first attempt to evaluate the thermodynamics of DNA TWJ formation by ITC.

— The interactions of cationic porphyrins with DNA oligonucleotides that form branched, three-way junction complexes (TWJ) were investigated using native gel electrophoresis, absorption spectroscopy and photochemical probing using DNA sequencing techniques. Meso-tetra(pa ra-N-trimethylaniliniumyrjporphine (TMAP), meso-tetra(4-JV-methylpyridiniumyl)porphine (T4MPyP) and meso-tetra(4-N-methylpyridiniumytyporphine (T3MPyP) were found to bind more tightly to DNA TWJ than to DNA duplexes. The binding to the junction DNA persists at high ionic strength, conditions that greatly decrease porphyrin binding affinity to duplex DNA. The TWJ DNA binding sites of TMAP and T4MPyP were localized to the junction region based on the observation of site- and structure-specific, porphyrin-sensitized photodamage to guanosine residues flanking the junction region.

The solution structure of a DNA three-way junction (TWJ) containing two unpaired thymidines was elucidated using two- and three-dimensional 1H nuclear magnetic resonance (NMR) spectroscopy. TWJs with unpaired nucleotides are ubiquitous structural motifs of complex single-stranded nucleic acids. In the presence of Mg2+, the TWJ complex adopts a unique conformation in which the bases of one of the oligonucleotides (“strand 1”) are continuously stacked across the junction. Guanosine 8 of strand 3 (S3-G8), which pairs with S1-C5. stacks on S2-G5, which is paired to S1-C6. The unpaired thymidine bases (S3-T6 and S3-T7) are exposed to the solvent, whereas the sugar of S3-G8 is largely buried. S3-T6 also interacts with the sugar residue of S3-G11. All three stems conform to B-type DNA.

A method has been found for reassembling fragment 1 of Escherichia coli 5S RNA from mixtures containing strand III (bases 69-87) and the complex consisting of strand II (bases 89-120) and strand IV (bases 1-11). The reassembled molecule is identical with unreconstituted fragment 1. With this technique, fragment 1 molecules have been constructed 15N-labeled either in strand III or in the strand II-strand IV complex. Spectroscopic data obtained with these partially labeled molecules show that the terminal helix of 5S RNA includes the GU and GC base pairs at positions 9 and 10 which the standard model for 5S secondary structure predicts [see Delihas, N., Anderson, J., & Singhal, R. P. (1984) Prog. Nucleic Acid Res. Mol. Biol. 31, 161-190] but that these base pairs are unstable both in the fragment and in native 5S RNA. The data also assign three resonances to the helix V region of the molecule (bases 70-77 and 99-106). None of these resonances has a "normal" chemical shift even though two of them correspond to AU or GU base pairs in the standard model. The implications of these findings for our understanding of the structure of 5S RNA and its complex with ribosomal protein L25 are discussed.

We have used several docking algorithms (GRAMM, FTDOCK, DOT, AUTODOCK) to examine protein-protein interactions between plastocyanin (Pc)/photosystem I (PSI) in the electron transfer reaction. Because of the large size and complexity of this system, it is faster and easier to use computer simulations than conduct x-ray crystallography or nuclear magnetic resonance experiments. The main criterion for complex selection was the distance between the copper ion of Pc and the P700 chlorophyll special pair. Additionally, the unique tyrosine residue (Tyr(12)) of the hydrophobic docking surface of Prochlorothrix hollandica Pc yields a specific interaction with the lumenal surface of PSI, thus providing the second constraint for the complex. The structure that corresponded best to our criteria was obtained by the GRAMM algorithm. In this structure, the solvent-exposed histidine that coordinates copper in Pc is at the van der Waals distance from the pair of stacked tryptophans that separate the chlorophylls from the solvent, yielding the shortest possible metal-to-metal distance. The unique tyrosine on the surface of the Prochlorothrix Pc hydrophobic patch also participates in a hydrogen bond with the conserved Asn(633) of the PSI PsaB polypeptide (numbering from the Synechococcus elongatus crystal structure). Free energy calculations for complex formation with wild-type Pc, as well as the hydrophobic patch Tyr(12)Gly and Pro(14)Leu Pc mutants, were carried out using a molecular mechanics Poisson-Boltzman, surface area approach (MM/PBSA). The results are in reasonable agreement with our experimental studies, suggesting that the obtained structure can serve as an adequate model for P. hollandica Pc-PSI complex that can be extended for the study of other cyanobacterial Pc/PSI reaction pairs.

We have refined the structure of the DNA Three-Way Junction complex, TWJ-TC, described in the companion paper by quantitative analysis of two 2D NOESY spectra (mixing times 60 and 200 ms) obtained in D2O solution. NOESY crosspeak intensities extracted from these spectra were used in two kinds of refinement procedure: 1) distance-restrained energy minimization (EM) and molecular dynamics (MD) and 2) full relaxation matrix back calculation refinement. The global geometry of the refined model is very similar to that of a published, preliminary model (Leontis, 1993). Two of the helical arms of the junction are stacked. These are Helix 1, defined by basepairs S1-G1/S3-C12 through S1-C5/S3-G8 and Helix 2, which comprises basepairs S1-C6/S2-G5 through S1-G10/S2-G1. The third helical arm (Helix 3), comprised of basepairs S2-C6/S3-G5 through S2-C10/S3-G1 extends almost perpendicularly from the axis defined by Helices 1 and 2. The bases S1-C5 and S1-C6 of Strand 1 are continuously stacked across the junction region. The conformation of this strand is close to that of B-form DNA along its entire length, including the S1-C5 to S1-C6 dinucleotide step at the junction. The two unpaired bases S3-T6 and S3-C7 lie outside of the junction along the minor groove of Helix 1 and largely exposed to solvent. Analysis of the refined structure reveals that the glycosidic bond of S3-T6 exists in the syn conformation, allowing the methyl group of this residue to contact the hydrophobic surface of the minor groove of Helix 1, at S3-G11. The helical parameters of the three helical arms of the structure exhibit only weak deviations from typical values for right-handed B-form DNA. Unusual dihedral angles are only observed for the sugarphosphate backbone joining the "hinge" residues, S2-G5 and S2-C6, and S3-G5 through S3-G8. The glycosidic bond of S3-G8also lies within the syn range, allowing favorable Watson-Crick base-pairing interactions with Si -C5. The stability of this structure was checked in 39 ps molecular dynamic simulation at 330 K in water. The structure of TWJ-TC retained the geometrical features mentioned above at the end of the simulation period. The final R(1/6)-factor of the refined structure is 5%.

The imino proton nuclear magnetic resonance spectrum of Escherichia coli 5S ribonucleic acid (RNA) changes when the Mg2+ ion concentration drops below physiological levels. The transition between the physiological and low magnesium spectral forms of 5S RNA has a midpoint at approximately 0.3 mM Mg2+. Many of the most conspicuous changes observed in the downfield spectrum of 5S RNA as the magnesium concentration is reduced are due to adjustments in the structures of helices I and IV and the disappearance of resonances originating in helix V. The binding of ribosomal protein L25 to 5S RNA in the absence of magnesium stabilizes helix V structures.

Publisher Summary This chapter describes the techniques used for purifying and manipulating 5S RNA and its various nucleolytic fragments, taking advantage of the availability of 5S RNA-overproducing strains. All the preparations described yield nuclear magnetic resonance (NMR)-scale quantities of end product. The fact that NMR is nondestructive, and that samples can be reused is helpful, but the need for large amounts of material remains. In this context, bacterial strains capable of overproducing the materials of interest are more than a mere convenience. The preparation of 5S RNA from chloramphenicol-treated pKK5-1- containing cells is straightforward. The cells are broken open; any of the standard methods will do. The ribosomes and cell debris are removed by centrifugation, and the RNA in the supernatant is isolated by phenol extraction. The 5S component of the resulting RNA mixture is purified by chromatography.